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| Status |
Public on Jun 18, 2021 |
| Title |
DKO0_Uhrf1_Input_rep1 |
| Sample type |
SRA |
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| Source name |
J1 mESC
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| Organism |
Mus musculus |
| Characteristics |
cell type: mESC
strain: J1
genotype: Dnmt3a + Dnmt3b KO
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| Treatment protocol |
N/A
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| Growth protocol |
mES cells were cultured in Knock Out DMEM media (Gibco) containing 15% FBS, 1% PEN/STREP, 1% glutamine, 1% NEAA, and 105 U LIF. For ES cell maintenance, dishes were coated with 0.2% gelatin and irradiated CD1 mouse embryonic fibroblasts (MEFs) were plated as a confluent layer of feeder cells. mES cells were seeded at a density of 50,000 cells per well of a six-well plate and were split every 3–4 days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
5 (histone) or 25 (Trim28) million cells were crosslinked with 1% FA for 5 or 8 minutes respectively at RT. Glycine was added to a final concentration of 125mM and mixed for 5 minutes at RT. Crosslinked cells were washed with DPBS 2x then spun down 3min 15000g. Cells were then incubated with 500µL cell lysis buffer (20 mM Tris-HCl ph8.0, 85 mM KCl, 0.5% NP 40) for 10 minutes on ice then spun down for 3min at 2500g. Supernatant was removed and the cell pellet was resuspended in 500µL of nuclear lysis buffer (10 mM Tris–HCl, pH 7.5, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) then incubated for 10 minutes on ice. Volume was increased to 1mL using Nuclei Lysis Buffer then sonicated on a Covaris E220 Evolution sonicator (PIP = 140.0, Duty Factor = 5.0, Cycles/Burst = 200, 10minutes).
After sonication, chromatin was spun down at 15000g for 10 minutes to pellet insoluble material. Volume was increased to 1.5mL with Chip Dilution Buffer (0.01% SDS, 1.1% Triton X-100,1.2mM EDTA, 16.7mM Tris-HCl pH 8.1, 167mM NaCl) and 2ug of H3K4me3 antibody (Abcam: ab8580), H3K36me3 (Active Motif 61101), H3K9me3 (Abcam, ab8898), or Trim28(Abcam, ab22553) were added. Immunoprecipitation mixture was allowed to rotate overnight at 4°C. The next day, 40µLs of Protein A Dynabeads (Thermo, 10001D) were added to the IP mixture and allowed to rotate for 4 hours at 4°C. This was followed by two washes of each: low salt wash buffer (0.1% SDS, 1% Triton X-100, 2mM EDTA, 20mM Tris-HCl pH 8.1, 150mM NaCl); high salt wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20mM Tris, pH 8.1, 500 mM NaCl); LiCl wash buffer (0.25M LCl, 1% NP40, 1% deoxycholate, 1mM EDTA, 10mM Tris-HCl pH 8.1); and TE buffer pH 8.0 (10mM Tris-HCl, pH 8.0, 1mM EDTA pH 8.0). DNA was eluted twice using 50µLs of elution buffer (0.5 to 1% SDS and 0.1 M NaHCO3) at 65°C for 15 minutes. 16µL of reverse crosslinking salt mixture (250 mM Tris-HCl, pH 6.5, 62.5 mM EDTA pH 8.0, 1.25 M NaCl, 5mg/ml Proteinase K) was added and samples were allowed to incubate at 65°C overnight. For library preparation, DNA was purified using AMPure XP beads (Beckman-Coulter) and treated with DNase-free RNase (Roche) for 30 minutes at 37 °C. DNA libraries were then end repaired and A-tailed using Ultra II End Repair/dA-Tailing Module (NEB) and adapters (Broad Institute, single index P7) were ligated using Blunt/TA Ligase Master Mix (NEB). Next, libraries were PCR amplified using Pfu Ultra II Fusion High-fidelity DNA Polymerase (Agilent) then size selected on a gel for fragments between 200-1000bp
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
ChIP-seq
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| Data processing |
The ChIPseq data as well as the control input sequencing were aligned to the mouse mm9 reference genome using BWA mem using the default parameter. Duplicate reads were removed using GATK MarkDuplicates (v4.1.0.0).
Peaks were called using the MACS2 peakcall (2.1.2_dev) function with default parameters.
Genome build: mm9
processed data files format and content: bigWig coverage files
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| Submission date |
Sep 23, 2020 |
| Last update date |
Jun 18, 2021 |
| Contact name |
Helene Kretzmer |
| E-mail(s) |
kretzmer@molgen.mpg.de
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| Organization name |
Max Planck Institute for Molecular Genetics
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| Department |
Genome Regulation
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| Lab |
Meissner Lab
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| Street address |
Ihnestraße 63
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| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE158460 |
Dnmt1 has global de novo methylation activity and is specifically targeted to transposable elements |
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| Relations |
| BioSample |
SAMN16249169 |
| SRA |
SRX9179205 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4801092_DKO0_Uhrf1_Input_rep1.bw |
164.0 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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